Intestinal protozoa are the cause of many human and animal diseases. When they infect domestic animals, severe economic losses may result. Intestinal protozoa of importance include cryptosporidium, trichomonads, histomonas, spironucleus, entamoeba, coccidia, toxoplasma and sarcocystis. One of the most problematic intestinal protozoa is Giardia.
Giardia lamblia is the most commonly found pathogenic parasite in western countries and is endemic in much of North America.sup.1. Giardia is a flagellated protozoan which is transmitted through the fecal-oral route.sup.2. The mechanism of pathogenesis in Giardia is poorly understood but it results in symptoms similar to many other gastrointestinal ailments. The most common symptoms are diarrhea, anorexia, malaise, abdominal distention and flatulence. Acute stages of the infection usually last only a few days although occasionally, especially in children, the chronic stage may last for months.sup.3.
Once inside its host, the Giardia trophozoites attach to the epithelium of host intestinal villi where they multiply, become encysted, and then are shed in the host feces. Some infected hosts become asymptomatic cyst passers after the short acute stage of the infection has passed.sup.4.
A major source of Giardia infection is contaminated drinking water. This has earned it the name "backpackers diarrhea" because of the large number of people infected due to exposure to contaminated water when camping.sup.4. Outbreaks of giardiasis are also a frequent problem in day care centers. Recurring Giardia infections in day care centers are common, and many of the children are found to be carriers of the infection although they show no symptoms. This makes detection and effective treatment of Giardia in day care centers difficult.sup.5.
A major area of interest in Giardia infections is the ability of animal hosts to act as reservoirs for human infective strains of Giardia. There is evidence that inter-species transmission of Giardia can and does occur. Human strains of Giardia have been shown to be infective in gerbils, mice, guinea pigs, raccoons, and beavers.sup.6-8. The common name given to Giardia infection in Canada, "beaver fever", was born when an epidemic of the infection resulted from the contamination of a water source by a family of three beavers.sup.9. Giardia infections are common in dogs and cats. It is estimated that 10-68% of dogs and 25% of cats are infected with Giardia. Studies of the prevalence of Giardia infection in domestic ruminants found infection in 17.7% of sheep and 10.4% of cattle, with the incidence being higher in lambs (35.6%) and calves (27.7%).sup.10.
As previously stated, clinical giardiasis may range from asymptomatic carriage of the parasite to an illness characterized by diarrhea, abdominal cramps, headache, gas, bloat, dehydration and malaise.sup.1,11. Weight loss and retardation of growth are also common problems associated with giardiasis in humans and animals.sup.11.
In humans, histological changes associated with Giardia include villus atrophy.sup.2. More recent studies using Mongolian gerbils as an animal model have shown diffuse shortening of the microvillus mainly in the duodenum as well as a decrease in the brush border enzyme activity. The enzyme deficiencies may be caused by the shortening of the epithelial microvilli.sup.12. Other studies have correlated Giardia infection with decreased weight gain, decreased food intake, decreased intestinal disaccharidase activities and villus atrophy.sup.13. The diffuse shortening of microvillus may be the result of a toxin produced by the Giardia.sup.12.
Given the prevalence of giardiasis and the difficulty in treating all those who are infected due to asymptomatic carriers, the development of a vaccine against Giardia is highly desirable. It has been shown that hosts will raise an immune response to Giardia and that they can retain long lasting immunity after the primary infection.sup.14,15. If a host is repeatedly exposed to Giardia, the risk of infection decreases.sup.16. Therefore, it is possible for a host to acquire immunity. The immune response of the host may explain the wide variability of host responses to Giardia. Sickness results when the immune system is unable to generate an adequate defense against the protozoan.sup.14. Previous studies in the area of Giardia immunity have been hampered by the lack of understanding of pathogenic mechanisms.
It has been demonstrated in natural and experimental Giardia lamblia and Giardia muris infections that both cellular and humoral immunity are generated by the host.sup.1,3,14,17,18. In natural and experimental infections, development of immunity has been associated with elimination of the parasite.sup.16,19. However, humans and animals with an elevated immune response to Giardia may still have clinical or subclinical giardiasis which may be due to inadequate immunity in the host.sup.14,20,21. It has been shown in humans and in animal models that severity of symptoms, course of infection, and infectivity rates are reduced following a second exposure to Giardia parasites.sup.14,16,19.
There have been numerous monoclonal and polyclonal antibodies produced in laboratory animals to Giardia lamblia and Giardia muris trophozoites and cysts. The purposes of producing these antibodies are for diagnostic purposes or reagents for laboratory studies. These antibodies are produced by employing standard hybridoma technology and using BALB/c mice. Polyclonal antibodies have been produced in rabbits and small rodents by performing multiple immunizations with antigens and Freund's adjuvant.
Passive immunization with anti-Giardia antibodies have been conducted. Butscher, et al..sup.22 demonstrated that intraperitoneal administration of monoclonal antibodies to a surface glycoprotein of G. muris trophozoites reduced parasitic burden in mice. Passive transfer of immunity has been demonstrated in mothers' milk.sup.23. Mother mice previously infected with Giardia muris were able to confer protection upon their suckling offspring while the milk of naive mothers was not protective.
There are extremely limited studies of active immunization of animals with Giardia trophozoites or with sub-unit vaccines. Roberts-Thomson et al. conducted a study where two strains of laboratory mice were vaccinated and challenged.sup.24. Intact Giardia muris trophozoites (10.sup.6) were combined with Freund's adjuvant (1:1 ratio) and injected intraperitoneally and in the footpad. Four weeks later animals were boosted in the same sites with the same dose without adjuvant. Control mice received only adjuvant or were untreated. One week later mice were challenged with Giardia muris cysts. Vaccinated BALB/c mice had a reduced cyst output for a shorter duration while there was no difference in the cyst output between control and vaccinated C3H/He mice. Thus, this study produced variable and ineffective results.
Vinayak et al..sup.11 isolated a 56 kDa protein from Giardia lamblia. Mice were subcutaneously immunized (Day 0) and orally immunized (day 7) with 100 .mu.g of multilammelar phosphatidylcholine liposomes (MPL)-entrapped 56 kDa surface associated antigen. Unimmunized and animals similarly immunized with MPL-entrapped PBS (Phosphate buffered saline) served as controls. All animals were challenged seven days after the last immunization dose. Immunization with MPL-entrapped 56 kDa surface-associated antigen resulted in a reduction in trophozoite colonization of the gut and duration of infection when compared to the control groups. It was suggested that the 56 kDa surface antigen immunoregulates the Giardia infection.
It is believed that Giardia secretes cytotoxins that influence the function or structure of the small intestinal mucosa.sup.25,26, but there has yet to be one or more toxic principles identified using classical methods for identification of cytotoxins.sup.25,26. Culture filtrates of Giardia have been shown to elaborate excretory/secretory products into the culture medium.sup.25. Culture filtrates have been shown to damage fibroblasts in culture as well as reduce salt and water absorption from perfused loops of rats but the role of these substances in the pathogenesis of giardiasis is unknown.sup.27.
There exists a need for an effective vaccine which can be used to prevent and treat protozoal infections, including giardiasis, in animals, including humans.